Project Summary/Abstract The overall hypothesis of this proposal is that HIV-1 assembly and release are temporally and spatially controlled processes. Further, we presume that regulated egress is critical for successful dissemination and thus pathogenicity of this virus. Our goal is to understand the molecular mechanisms that control viral release and transmission to target cells. Knowledge, at the cellular level, about this late step of the viral replication cycle will enable the development of strategies aimed at interfering with HIV-1 spread in infected individuals. The molecular composition of the membrane segments through which HIV-1 buds remains poorly characterized. During the previous funding cycle we demonstrated that the membrane microdomains through which HIV-1 exits are enriched in tetraspanins. Tetraspanins are cellular proteins implicated in various membrane-based processes including cell migration, cell-cell fusion, and signaling at immunological synapses. A significant proportion of the proposed work will be dedicated to answer the question if tetraspanins play functional roles in the targeting of HIV-1 assembly/release. The specific aims of this proposal are: 1 To further characterize the formation, composition and turnover of the plasma membrane microdomains through which HIV-1 buds. 2 To evaluate if tetraspanins advance the formation of the virological synapse by regulating the targeting of HIV-1 components to these sites 3 To test the hypothesis that tetraspanins activities at established virological synapses are necessary for efficient transmission of HIV-1 particles.